Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery
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Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery

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Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery

Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery

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    Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery Presentation Transcript

    • The Latest Discovery: Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery Dr Sarah Jones Molecular Pharmacology Research Group, University of Wolverhampton
    • Overview  History and Background of CPPs  Hurdles and Progress  Where the field is at the moment  Targeting Protein-Protein Interactions, from Bioportides to Sperm
    • Cell Penetrating Peptides (CPPs) • Inert vectors for the delivery of bioactive cargoes into the intracellular milieu • Intracellular delivery of Peptides, Proteins, Drugs, Oligonucleotides (siRNA, PNA), Plasmids • Viable alternative to viral vectors and current non-viral intracellular delivery
    • CPP History: A Long Time Coming!  1988 - The TransActivator of Transcription protein (TAT) derived from the HIV virus entered cells  1994- Penetratin –Helix 3 of the Antennapedia homeodomain  1997 - Tat48-60, a truncated arginine-rich sequence conferred cellular penetration. Tat AntHD43-58 Penetratin
    • Classification of CPPs Protein-derived Chimeric Penetratin Tat48-60 pVEC RQIKIWFQNRRMKWKK-NH2 Transportan GWTLNSAGYLLGKaINLKALAALAKKIL-NH2 Transportan 10 GRKKRRQRRRPPQ LLIILRRRIRKQAHAHSK AGYLLGKaINLKALAALAKKIL-NH2 PepFects Synthetic/Designed Polyarginine RRRRRRRR (RXR)4 (R(Ahx)R)4 CADY Ac-GLWRALWRLLRSLWRLLWRA-Cya
    • Most CPP are polycationic sequences Primary Sequence Name/Source HIV-1 Tat-derived peptides GRKKRRQRRRPPQ QPPRRRQRRKKRG Tat48-60 RI-Tat Penetratins RQIKIWFQNRRMKWKK RRRRRRRQIKIWFQNRRMKWKK AntHD43-58 (Penetratin) R6-Penetratin Other arginine-rich peptides RRRRRRRRR (R(Ahx)R)4 R8 and R9 are the most common (RXR)4 Predominantly amphipathic sequences AGYLLGKINLKALAALAKKIL INLKKLAKL(Aib)KKIL LLIILRRRIRKQAHAHSK Transportan10 Mitoparan pVec Hydrophobic Sequences CSIPPEVKFNKPFVYLI C105Y
    • Uptake mechanisms, a fixation! Direct membrane translocation (4oC) Fixation artifacts (Live cell imaging) Endocytosis (37oC, ATP) Clathrin-mediated Caveolin-mediated Macropinocytosis Dependent upon sequence, cell type, concentration and cargo (biochemical properties and size)
    • To Endocytose or not to Endocytose? That is the question Mishra A et al. Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions PNAS (2011) 108 16883
    • Progress in CPP Technologies PepFect Technologies AGYLLGKINLKALAALAKKIL-NH2 TP10 PepFect3 Stearyl-AGYLLGKINLKALAALAKKIL-NH2 PepFect6 Stearyl-AGYLLGKINLKALAALAKKIL-NH2 PepFect14 Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2 • Oligonucleotide therapeutics-siRNA and splice correcting oligonucleotides • Ecsapes endosomal entrapment associated with the delivery of larger cargoes Ülo Langel Stockholm University CPP Technologies in Clinical Development • PsorBan (CellGate) – heptaarginine coupled to cyclsporin A (psoriasis) • KAI-9803 (Kai Pharmaceuticals) - tat coupled to a peptide inhibitor of PKCd (reperfusion injury) • XG-102 (Auris Medical)– tat coupled to a JNK inhibiting peptide (traumatic hearing loss)
    • What Cell Penetrating Peptides DO NOT DO! The Lipinski Rule of Five •Its molecular weight is less than 500. •The compound's lipophilicity, expressed as a quantity known as logP (the logarithm of the partition coefficient between water and 1-octanol), is less than 5. •The number of groups in the molecule that can donate hydrogen atoms to hydrogen bonds (usually the sum of hydroxyl and amine groups in a drug molecule) is less than 5 •The number of groups that can accept hydrogen atoms to form hydrogen bonds (estimated by the sum of oxygen and nitrogen atoms) is less than 10.
    • Bioactive Cell Penetrating Peptides (Bioportides) Enhance the Repertoire of Druggable Targets Expansion of Druggable Targets It is estimated that 8-10% of the human genome encodes diseasemodifying proteins Only 10% of the druggable genome can be targeted by conventional approaches (SMDs) New Chemical Entities which target Protein-Protein Interactions are gaining momentum as an attractive therapeutic modality Proteomics and interactomics will identify many other PPIs that can be modulated by peptides Intracellular Drug Target Space
    • Mast cell secretion Activation of p42/44 MAPK
    • Sychnologic CPP Rhegnylogic CPP Sequence Target Activity Nosangiotide ND Anti-angiogenic Sequence Target Activity Pathology Camptide G proteins cAMP modulation Tat-DI1 Raf dimerizatio n Inhibits proliferation of NSCLC cell lines Cancer Cyt c77-101 ER Apoptogenic BIP BAX Antp-MEK1 ERK Inhibition of ERK2 activation Cancer Anti-apoptogenic (neuroprotection) Tat48-60-P10 PCNA Apoptogenic Cancer Mouse PrP1-28 Prion Proteins Anti-prion infection STAT-6-IP STAT-6 Inhibitor of TH2 cytokine production Allergic airways disease AT1AR304-318 G proteins Blood vessel contraction Hph-1ctCTLA-4 TcR signalling Inhibition of TcR signalling. Reduction in Th2 cytokines, serum IgE Allergic airways disease Vasostatin-1 Heparan Sulphate Proteoglycans eNOS activation (vasodilatory) Arf (1-22) ND Apoptogenic Mitoparan VDAC (mitochondria) Apoptogenic Stapled p53derived peptides p53-hDM2 Reactivation of p53 apoptosis pathway Tat-acpTrkA666-676 Nup153-Cyt TrkA activation loop TrkA antagonism NPC Apoptogenic Inflammator y pain Cancer
    • Bioactive Cell Penetrating Peptides - Bioportides Signal Transduction Domain Position 85 IKKKEERADLIAYLKK 2 16 86 KKKEERADLIAYLKKA 3 17 84 GIKKKEERADLIAYLKK 3 17 85 IKKKEERADLIAYLKKA 3 17 QSAR Prediction Algorithm No. of Amino Acids 16 Whole Protein Sequence 86 KKKEERADLIAYLKKAT 2 Prediction algorithm: Hällbrink et al. Int. J. Peptide Res. Ther. 11, 249 (2005) CPP mimic of GPCRs: Ostlund et al. Int. J. Peptide Res. Ther. 11, 237 (2005) CPP Probability
    • Anti-angiogenic Properties of Nosangiotide (eNOS492-507) A Bioportide Derived from Endothelial Nitric Oxide Synthase Nosangiotide is located within the a-helix domain (shown in yellow) that tightly binds calmodulin. Diagram adapted from Aoyagi et al., EMBO J. 22, 766-775 (2003) Sequence RKKTFKEVANAVKISASLMG RKKTFKEVANA RKKTFKEVANAVK RKKTFKEVANAVKI RKKTFKEVANAVKISA Early distribution Cells were incubated for 45 mins with rhoeNOS492-507 (5mM) in endothelial cell medium plus growth supplement then transferred to DMEM w/o phenol red for confocal visualization CPP Index Later nuclear and cytoplasmic distribution 1 3 3 3 3 Nosangiotide Cells were incubated for 80 mins with rhoeNOS492-507 (5mM) in endothelial cell medium plus growth supplement then transferred to DMEM w/o phenol red for confocal visualization
    • Nosangiotide (eNOS492-507) Inhibits Biological Features of FGF-induced Angiogenesis in vitro (a) (b) (c) 250 1750 90,000 80,000 70,000 mean of closed areas per field number of migrated cells cell number 1500 1250 1000 200 150 100 750 60,000 500 -9 -8 -7 492-507 log { [eNOS -6 ] (M) } Proliferation IC50 = 83.7 nM -5 -8 -7 log { [eNOS -6 492-507 Migration IC50 = 38.2 nM -5 ] (M) } -4 50 -9 -8 -7 -6 492-507 log { [eNOS -5 -4 -3 ] (M) } Tube Formation IC50 = 509 nM Nosangiotide is a potent inhibitor of FGF-2 (25 ng/ml)-induced proliferation (a), migration (b) and tube formation (c) of primary endothelial cells
    • Nosangiotide inhibits FGF-induced angiogenesis A) Carrier control B) FGF-2 (200ng) C) Nosangiotide (0.5 nmole) D) FGF-2 plus nosangiotide (0.5 nmole) E) FGF-2 with nosangiotide (0.05 nmole) F) FGF-2 with nosangiotide (0.005 nmole). Howl et al. (2012) Cell. Mol. Life Sci. 69, 2951 .
    • Identify Bioportide Binding Partners Future Work Stapled Peptides Biotinylated CPP as Molecular Fishing Rods C-terminal helix N-terminal helix Biotinylated CPP Primary Sequence Position KGKKIF KGKKIFI GKKIFIMK KGKKIFIM KGKKIFIMK EKGKKIFIMK Swiss 3T3 Streptavidin-coated multiwell plates Primary Sequence 5-10 5-11 6-13 5-12 5-13 4-13 Position VGIKKK KMIFVGIKKK KMIFVGIKKKEERA KKKEERADLIAYLKKA GIKKKEERADLIAYLKK IKKKEERADLIAYLKKA VGIKKKEERADLIAYLKK 83-100 GIKKKEERADLIAYLKKA FVGIKKKEERADLIAYLKK 82-100 VGIKKKEERADLIAYLKKA IFVGIKKKEERADLIAYLKK 81-100 FVGIKKKEERADLIAYLKKA KMIFVGIKKKEERADLIAYLK 79-99 MIFVGIKKKEERADLIAYLKK 80-100 IFVGIKKKEERADLIAYLKKA KMIFVGIKKKEERADLIAYLKK 79-100 MIFVGIKKKEERADLIAYLKKA TKMIFVGIKKKEERADLIAYLKK 78-100 KMIFVGIKKKEERADLIAYLKKA79-101 GTKMIFVGIKKKEERADLIAYLKKA TKMIFVGIKKKEERADLIAYLKKA KMIFVGIKKKEERADLIAYLKKAT GTKMIFVGIKKKEERADLIAYLKKA77-101 TKMIFVGIKKKEERADLIAYLKKAT KMIFVGIKKKEERADLIAYLKKATN 83-88 79-88 79-92 86-101 84-100 85-101 84-101 83-101 82-101 81-101 80-101 77-101 78-101 79-102 78-102 79-103  Cross linking of peptide side chains TRYPSIN DIGEST  Metabolic stability  Enhance propensity for cellular penetration Dr Ashley Martin MALDI TOF/TOF Proteomics Unit Cancer Studies University of Birmingham, UK a-aminoisobutyric acid
    • LRRK2-derived Bioportides Highly Probable CPPs collectively spanning the entire LRRK2 protein CPPs containing mutations known to cause Parkinson’s disease Sequence Position Sequence Position (mutation) TLKKLIVRLNNVQEGKQI 15-32 KKQCFKNDIHKLVLAALNRFI 534-554 (K544E) RKILLSKGIHLN 433-444 SPKLVELLLNSGSREQDVRKAL 754-775 (P755L) KLVLAALNRFIG 544-555 QIISLLLRRLALDVANNSICLG 785-806 (R793M) RKALTISIGKGDSQIISLLLRR 772-793 NLRKQTNIASTLARMVIRYQMK 828-849 GNKISGICSPLRLKELKILNL 1116-1136 (I1122V) LLKRKRKILSSDDSLRSSKLQ 945-965 ATVGIDVKDWPIQIRDKRKRDL 1367-1388 (I1371V) SPLRLKELKILNLSKN 1124-1139 LISLLAAGIRPRMLVMELASKG 1932-1953 (R1941H) KKTEKLCGLIDCVHFLREVMVK 2378-2399 (G2385R) HIGCKAKDIIRFLQQRLKKAVP 1310-1331 LQQRLKKAVPYNRMKLMIV 1322-1340 GSGKTTLLQQLMKTKK 1344-1359 ATVGIDVKDWPIQIRDKRKR 1367-1386 KQRKACMSKITKELLNKRGFPA 1460-1481 LAKLRKTIINESLNFKIRDQLV 1497-1518 PVIDRKRLLQLVRENQLQL 1546-1564 AVKIFNKHTSLRLLRQ 1904-1919 LISLLAAGIRPR 1932-1943 RLLQQDKASLTRTLQHRIA 1957-1975 LTRRILLPKNVI 2140-2151 KTVKLKGAAPLKILNIGN 2278-2295 RVKTLCLQKNTALWI 2413-2427 LLLDLSTRRLIRVI 2435-2448 IEVRKELAEKMRRT 2511-2524 > 5000 Probable CPP
    • LRRK2: Not just Parkinsons!  LRRK2 role in Inflammatory Bowel Disease (Liu, Z. et al., (2011) Nature Immunology 12,10631070) LRRK2  LRRK2 sequesters NFAT in the cytoplasm  LRRK2 deficiency enhances susceptibility to experimental colitis and enhances nuclear localization of NFAT  NFAT Luciferase reporter assay (Armesilla, A.L. et al. (1999) Mol. Cell. Biol. 19, 2032-2043)  Bioportides facilitate relocation of NFAT from the cytoplasm to the nucleus?  Bioportides abrogate PMA and Ca2+ ionophore induced relocation of NFAT?  The role of the Immune System in PD
    • Candidate Bioportides for Modulation of NFAT Translocation LRRK21310-1331 T2397M LRRK21124-1139 LRRK215-32 1 ARM LRRK21116-1136 I1122V ANK LRR LRRK22378-2399 LRRK21367-1386 ROC G2385R COR Kinase WD40 Leucine-Rich Repeat domain KLEQLILEGNKISGICSPLRLK GNKISGICSPLRLKELKILNL LRRK21116-1136 GNKISGVCSPLRLKELKILNL LRRK21116-1136 (I1122V) SPLRLKELKILNLSKN LRRK21124-1139 LRRK21310-1331 (C-terminal helix) Inhibits NFAT translocation 2527 Van Craenenbroeck et al., (2012) Purification and preliminary biochemical and structural characterisation of the leucine rich repeat domain of LRRK2. Biochem. Biophys. Acta 1824, 450 WT inhibits NFAT translocation Mutant enhances NFAT translocation Enhances NFAT translocation HIGCKAKDIIRFLQQRLKKAVP
    • LRRK22378-2399 includes the very common T2397M risk allele for Crohn’s Disease LRRK21310-1331 T2397M LRRK21124-1139 LRRK215-32 1 ARM LRRK21116-1136 I1122V ANK LRRK2-derived bioportides modulate NFAT translocation. HEK293 cells were transiently transfected with a luciferase reporter vector (pNFAT-Ta-Luc; Clontech). Luciferase activity was measured as an indicator of NFAT translocation to the nucleus. Cells were stimulated with PMA (20 ng/ml) and the calcium ionophore A23187 (1 mM) for 16 hours. Luciferase activity was calculated as fold induction over the value of the reporter vector in un-stimulated cells. LRR LRRK22378-2399 LRRK21367-1386 ROC COR Kinase G2385R 2527 WD40
    • Building Cell Selectivity into CPP Delivery • CPP can be readily incorporated into multimeric complexes • An inherent lack of specificity can be surmounted by the inclusion of tissue specific targeting peptides • Phage Display Technology has generated an abundance of tissue homing peptides
    • Building Cell Selectivity into CPP Delivery Homing Peptides CPP Sequence
    • Peptide-Based Glioma-Targeted Drug Delivery Vector gHoPe2 Coronal section of a mouse brain with U87 tumor in the right striatum. The tumour area is circled with a dotted line. (B) H&E stained hemisphere of brain and glioma. (C) The animals received an i.v. injection of FAM-labeled gHoPe2 3 h before tissue collection Liver Kidney Intracranial Tumour Model Intact brain Intracranial tumour FAM-gHo Eriste, E., Kurrikoff, K., Suhorutsenko, J., et al. Bioconjugate Chemistry (2013). In press Scale 100 μm. FAM-gHoPe2
    • Building Cell Selectivity into CPP Delivery Activatable CPPs Exploitation of tissue specific endopeptidases cargo ++++++++++++++ cargo ++++++++++++++ -----------Cellular internalization impaired Cellular internalization MMP2 Matthias Hallbrink – NoPe (YTA4 + MMP-2 cleavage site + inactivationg region) -tumour imaging in vivo.
    • The Challenge: Penetrating the Impenetrable Internal Volume Nucleus • • • Is reduced. Mature sperm lacks a variety of organelles: endoplasmic reticulum, Golgi apparatus, cytosolic ribosomes. • Once Spermatozoa are released into the seminiferous tubules, genomic transcription and translation have largely been silenced. Conventional molecular biology techniques are thus redundant. Modulation of sperm cell biology is restricted to cell permeable agents. Plasma Membrane • • • • Lipid composition of plasma membrane is highly polarized and compartmentalized. It appears to be incapable of endocytotic events. Static physical barrier Detergents are detrimental to protein function http://www.flickr.com/photos/wellcomeimages/5814253423/
    • Understanding Sperm Biology with CPP Sperm are transcriptionally and translationary inactive Studies of their intracellular biology are restricted to cell permeable agents CPP are ideal vehicles for the study of sperm intracellular biology, from motility to capacitation and the acrosome reaction Can be easily isolated from bulls semen with supplemented EBSS containing 0.3% BSA (“swim up method”) sEBSS (0.3% BSA) semen
    • Differential Intracellular Distribution of CPP 6 5 4 3 2 1 13 21 iM rV 1a R 10 P 0 Ta t 10 5Y M itP TP 10 iM it P Rho-C105Y (5mM) 7 C Rho-tat (5mM) + DIC 8 Rho-C105Y (5mM) tr at in Rho-penetratin (5mM) Mitotracker (500nM) Rho-tat (5mM) + DIC C105Y Fluorescence minus background (A.U.) Rho-penetratin (5mM) Tat Pe ne Penetratin Comparative analysis of CPP translocation efficacies into bovine spermatozoa. Spermatozoa were incubated with TAMRAlabelled CPP (5 mM) for 1 h at 37 oC. Data are mean + S.E.M. from 3 experiments performed in triplicate.
    • The Mitochondrial-localising CPP Mitoparan Rho-MitP (5mM) Rho-MitP (5mM) Mitotracker (500 nM) Mitotracker (500 nM) merge Mitochondrial Midpiecei Rho-TP10 (5mM) Mitotracker (500 nM) merge
    • . Protein Delivery Rho-TP10 (3 mM) + avidin Alexa Fluor® 488 (1 mM) Spermatozoa Merge DIC Avidin Merge Swiss 3T3 TP10 Swiss 3T3 Rho-TP10 (1 mM) + avidin Alexa Fluor® 488 (0.33 mM) Rho-TP10 (3 mM) + avidin Alexa Fluor® 488 (1 mM) Merge Avidin Merge Avidin TP10 Merge DIC TP10 Merge DIC
    • Why are CPP unable to deliver large proteins into sperm? Translocation Kinetics of C105Y Endocytosis Incompetent Spermatozoa Swiss 3T3 Swiss 3T3 Spermatozoa Spermatozoa Transferrin Alexa Fluor® 488 (50 mg/ml) Transferrin Texas Red® (50 mg/ml) LysoTracker® Red (75 nM)) LysoTracker® Red (75 nM)) Dextran Texas Red® (10 mM) Dextran Texas Red® (10 mM) 10 8 t0.5 = 0.70 min 6 C105Y tat 4 rV1aR102-113 2 0 0 10 20 30 40 50 60 Fluorescence minus background (A.U.) Fluorescence minus background (A.U.) 10 12 8 6 t0.5 = 7.02 min rV1aR102-113 2 0 0 10 20 30 40 50 60 Time (min) Time (min) Internalisation occurred with first order saturable kinetics (F = Fmax x t/t0.5 + t, GraphPad Prism 5). C105Y tat 4 Cells were incubated at 37oC with TAMRA-labelled peptides (5mM) for the times indicated. Normalised data (compared to tat-assigned a value of 1) are expressed as mean fluorescence (minus background) + s.e.m. from 3 experiments performed in triplicate. Direct membrane translocation is the sole mechanism of CPP import into sperm
    • Bioportides as Modulators of Human Sperm [Ca2+]i Signalling STIM1 ORAI1 Activating Region 442 334 EEELE CC1 (248-342) CC3 ? STIM1 (485685) KIKKK CC2 CC2 CC2 EEEL E +++ CC1 CC3 (364-388) (399-432) + --- Occluded SOAR CC1 KIKKK CC3 ? STIM1 (485685) Free SOAR Orai1 Plasma membrane  Progesterone-the best-characterised agonist of human sperm [Ca2+]i signalling.  Motility and the acrosome reaction  Biphasic [Ca2+]i response Store depletion Endoplasmic reticulum STIM1 STIM1371-392 H-KQLLVAKEGAEKIKKKRNTLFG-NH2 Scr- STIM1371-392 H-LKNKFKGVKLAEIEKQALKGTR-NH2
    • A 140 B 120 100 80 60 Non-capacitating MitP nosangiotide tat 40 20 0 0 20 40 60 80 100 120 140 160 180 120 100 80 60 Capacitating camptide 40 Cyt c 5-13 C105Y 20 0 0 20 40 60 80 100 120 140 160 180 C 100 90 % cell viability 140 Time (min) Time (min) 110 % motile cells (*rapid) relative to controls % motile cells (*rapid) relative to controls CPP Import is Compatible with Sperm Motility and Viability A, B. Motility data were collected from human sperm cells treated with 5 mM CPP. 80 C105Y MitP tat iMitP iMP penetratin nosangiotide 70 60 50 40 30 Each peptide was tested on samples from 3 individual donors. Data are shown as % rapid cells from treated samples relative to that of controls and expressed as mean + s.e.m. *rapid cells = velocity (average path) ≥25 µms-1 and straightness ≥ 80%. C. Isolated bovine spermatozoa were treated with CPP for 1 h at the concentrations 20 indicated. Cell viability was measured by MTS conversion and expressed as a 10 percentage of those spermatozoa treated with vehicle alone (sEBSS). 0 -6.0 -5.5 -5.0 -4.5 log { [peptide] (M) }
    • Conclusions  CPPs for site-specific delivery of bioactive cargoes into mammalian sperm  Cargo size is critical  CPP technologies as valuable tools for the investigation and modulation of fundamental processes of sperm physiology such as maturation, capacitation, motility, hyperactivation and fertilisation. Pantechnia Jones, S., Lukanowska, L., Suhorutsenko, J., Oxenham, S., Barratt, C., Publicover, S., Copolovici, D.M., Langel, Ü. and Howl, J. (2013) Intracellular translocation of cell penetrating peptides into spermatozoa. Human Reproduction DOI: 10.1093/humrep/def064.
    • The Future for CPP Therapeutics Modifications to enhance stability are now surmountable Routes of administration Mechanisms for moving forward Marcus Evans Discovery and Evolution Summits Mass Screening, Formulation and Analogues
    • Acknowledgements Scientific contributors University of Wolverhampton, UK John Howl Monika Lukanowska University of Birmingham, UK Michelle Farquhar Ashley Martin Steve Publicover MRC Protein Phosphorylation Unit, University of Dundee, UK Dario Alessi University of Dundee, UK Chris Barratt Senga Oxenham Department of Neurochemistry, Stockholm University, Sweden University of Tartu, Estonia Ulo Langel University of Manchester, UK Shant Kumar